JPWO2006080420A1 - Antigen-specific T cell inducer, antibody production inducer, immune enhancer, and vaccine - Google Patents

Abstract

A novel antigen-specific T cell inducer, a novel antibody production inducer, a novel immune enhancer, and a novel vaccine, and a novel antigen-specific T cell induction method, antibody production induction method, a novel immune enhancement method, and a novel tumor treatment Methods and novel pathogen treatment methods are provided. A complete adjuvant prepared by inactivating herpes simplex virus (HSV) and adding it to an incomplete adjuvant is used as an antigen-specific T cell inducer, antibody production inducer, or immune enhancer, These antigen-specific T cell inducers, antibody production inducers, or immunopotentiators are used in combination with antigens to form vaccines. At that time, by using a cancer-specific antigen or a pathogen-specific antigen as an antigen, a tumor treatment method and a pathogen infection treatment method are obtained.

Description

  The present invention relates to an antigen-specific T cell inducer, an antibody production inducer, an immunopotentiator, and a vaccine containing herpes simplex virus (HSV).

  When inoculating a substance (antigen) that causes an immune reaction in a living body, in order to enhance the immune reaction, the antigen is mixed with an immunopotentiator called an adjuvant and inoculated. As adjuvants, Freund Complete Adjuvant and Freund Incomplete Adjuvant, which are often used in animal experiments (Broderson, JA, Lab. Anim. Sci. 39 (1989), 400-406). ), Various things are known. For example, Freund's complete adjuvant is an emulsion containing mycobacteria heat-sterilized in mineral oil, and Freund's incomplete adjuvant is an emulsion in which Freund's complete adjuvant is not added with mycobacteria. In Freund's complete adjuvant, mycobacteria are active ingredients having immune enhancing activity.

  When administering a vaccine to an animal, an adjuvant is added to enhance the effect of the vaccine. There are live vaccines, inactivated vaccines, component vaccines, etc., but adjuvants are added to inactivated vaccines and component vaccines in particular, and antibodies are effectively produced even when antigens with weak immunogenicity are used. Will be able to.

  In recent years, new types of adjuvants have been developed as new vaccines such as component vaccines and cancer vaccines have been developed, and the importance of adjuvants has increased clinically (Jin H, et al., Vaccine 22 (2004), 2925-2935; Flarend, R et al., Vaccine 15 (1997), 1314-1318; Villacres-Eriksson M, et al., Cytokine 9 (1997), 73-82; Weeratna, R, et al., Vaccine 18 (2000), 1755-1762).

  The present invention was made for the purpose of developing a novel antigen-specific T cell inducer, a novel immune enhancer, a novel antibody production inducer, and a novel vaccine against the background of the above-described technology.

  The antigen-specific T cell inducer according to the present invention is an antigen-specific T cell inducer for antigens other than HSV, containing herpes simplex virus (HSV) as an active ingredient. Accordingly, a method for inducing the antigen-specific T cell against an antigen other than HSV administered to humans and non-human vertebrates, wherein the antigen-specific T cell induction is used in combination with the antigen. A method characterized by administering an agent also belongs to the technical scope of the present invention. This antigen-specific T cell inducer is preferably an antigen-specific cytotoxic T cell inducer. The herpes simplex virus is preferably inactivated, and may be inactivated by, for example, heating, UV irradiation, radiation irradiation, fixation, or genetic recombination. Furthermore, the antigen-specific T cell inducer may contain incomplete Freund adjuvant (IFA).

  The antibody production inducer according to the present invention is an antibody production inducer against an antigen other than HSV, which contains herpes simplex virus (HSV) as an active ingredient. Therefore, a method of inducing antibody production against an antigen other than HSV, administered to humans and non-human vertebrates, characterized in that the antibody production inducer is administered in combination with the antigen. This method also belongs to the technical scope of the present invention. The herpes simplex virus is preferably inactivated, and may be inactivated by, for example, heating, UV irradiation, radiation irradiation, fixation, or genetic recombination. Furthermore, the antibody production inducer may contain an incomplete Freund adjuvant (IFA).

  The immunopotentiator according to the present invention is an immunopotentiator for antigens other than HSV, which contains herpes simplex virus (HSV) as an active ingredient. Therefore, a method for enhancing immunity against antigens other than HSV in humans and non-human vertebrates, which comprises administering the immunopotentiator, also belongs to the technical scope of the present invention. The herpes simplex virus is preferably inactivated, and may be inactivated by, for example, heating, UV irradiation, radiation irradiation, fixation, or genetic recombination. Furthermore, the immune enhancing agent may contain incomplete Freund's adjuvant. Here, an immune enhancer generally refers to a drug that enhances an immune response in a living body. This immune reaction includes antigen-specific T cell induction, antibody production induction, natural killer cell activation induction, dendritic cell activation induction, inhibitory T cell induction inhibition, and the like.

The vaccine according to the present invention is a vaccine against an antigen other than herpes simplex virus (HSV), and is a vaccine containing an immunopotentiator containing herpes simplex virus as an active ingredient and the antigen. The vaccine may be a cancer vaccine or a vaccine against pathogen infection. Therefore, a method for treating a tumor in humans and non-human vertebrates or a method for treating pathogen infection, which is characterized by inoculating the vaccine, also belongs to the technical scope of the present invention. The herpes simplex virus is preferably inactivated, and may be inactivated by, for example, heating, UV irradiation, irradiation, fixation, or genetic recombination. Furthermore, the vaccine may contain incomplete Freund's adjuvant. The treatment method referred to herein includes not only a disease treatment method but also a prevention method before the disease.

-Cross reference with related literature-
This application claims the benefit of priority of Japanese Patent Application No. 2005-20393 filed on Jan. 27, 2005, and is incorporated herein by reference.

In one embodiment of the present invention, GARC-1 peptides (mutant and wild type) of T cells obtained from mice administered with a vaccine comprising GARC-1 mutant peptide and inactivated HSV are presented. It is the graph which represented the reactivity with respect to a cell with the production amount of IFN-gamma. In one embodiment of the present invention, the reactivity of T cells obtained from a mouse administered with a vaccine containing GARC-1 mutant peptide and inactivated HSV to GL261 cells is expressed as IFN-γ production. It is a represented graph. In one embodiment of the present invention, the reactivity of CD8 positive T cells obtained from a mouse administered with a vaccine containing a GARC-1 mutant peptide and inactivated HSV to GL261 cells was determined as the production of IFN-γ. It is a graph expressed in quantity. In one embodiment of the present invention, (A) and (B) show the volume of tumor cells transplanted into a mouse in which tumor cells were transplanted and administered a vaccine comprising AH1 peptide and inactivated HSV. (C) is a diagram evaluating the cytotoxic activity of CD8 positive T cells obtained from the mice against CT26 cells, (D) FIG. 2 is a graph showing the amount of IFN-γ produced in response to AH1 antigen of CD4 positive T cells and CD8 positive T cells obtained from a mouse administered with a vaccine containing AH1 peptide and inactivated HSV. is there. In one embodiment of the present invention, in a mouse administered with a vaccine containing influenza virus HA protein and inactivated HSV, (A) shows ^Kd- restricted HA of CD8-positive T cells obtained from the mouse. peptide is a diagram showing an evaluation of the cytotoxic activity of the reaction recognizes the (MHC class I), (B) is a CD4-positive T cells obtained from the mice, ^{I-E d} restricted HA peptide (MHC class It is the figure which evaluated the proliferative activity which recognizes and reacts II).

  Unless otherwise stated in the embodiments and examples, J. Sambrook, EF Fritsch & T. Maniatis (Ed.), Molecular cloning, a laboratory manual (3rd edition), Cold Spring Harbor Press, Cold Spring Harbor, New York (2001); FM Ausubel, R. Brent, RE Kingston, DD Moore, JG Seidman, JA Smith, K. Struhl (Ed.), Standard Protocols in Molecular Biology, John Wiley & Sons Ltd. The method described in the protocol collection, or a modified or modified method thereof is used. In addition, when using commercially available reagent kits and measuring devices, unless otherwise explained, protocols attached to them are used.

  The objects, features, advantages, and ideas of the present invention will be apparent to those skilled in the art from the description of the present specification, and those skilled in the art can easily reproduce the present invention from the description of the present specification. it can. The embodiments and specific examples of the invention described below show preferred embodiments of the present invention, and are shown for illustration or explanation. It is not limited. It will be apparent to those skilled in the art that various modifications and variations can be made based on the description of the present specification within the spirit and scope of the present invention disclosed herein.

== Preparation of antigen-specific T cell inducer, antibody production inducer, immunopotentiator and antigen mixture ==
The type of HSV contained in the antigen-specific T cell inducer, antibody production inducer, immunopotentiator or vaccine according to the present invention is not particularly limited, but in the examples, a KOS strain, which is a type I wild strain, was used. . HSV can be grown according to a conventional method using Vero cells or the like.

The HSV used in the present invention is preferably inactivated by heating, UV irradiation, radiation irradiation, fixation, gene recombination or the like so as not to grow in vivo. Hereinafter, as typical conditions for each operation, when heating the HSV, for example, the treatment is performed at 56 ° C. for about 30 minutes. In the case of UV irradiation, UV lamps (wavelength, 253.7 nm) are irradiated at a distance of 10 to 15 cm and a total of 120 to 650 J / m ² (De Roda Husman AM,. Et al., Appl Environ Microbiol. 70 (2004) : 5089-5093.). In the case of irradiation, a total of 200-800 Gy is applied at 0.96 Gy / s (De Roda Husman AM,. Et al., Appl Environ Microbiol. 70 (2004): 5089-5093). Moreover, what is necessary is just to use well-known tissue fixing agents, such as a formaldehyde solution, formalin, and glutaraldehyde, when fixing. Inactivation by genetic recombination is, for example, controlling viral replication by genetic manipulation (by performing genetic recombination so that the viral genome is replicated only in tumor cells and not in normal cells) (Mineta T, et al. Attenuated multi-mutated herpes simplex virus-1 for the treatment of malignant gliomas. Nat Med. 9 (1995), 938-943.), Or to prevent the virus from replicating by genetic manipulation (Lim F, et al. Biotechniques. 20 (1996): 460-469.). In addition, the said conditions are typical example conditions, In specific operation, you may not be in the above-mentioned conditions and may not be inactivated depending on the case.

  Such HSV is used as an antigen-specific T cell inducer, antibody production inducer, or immune enhancer, and mixed with an antigen that is the target of antigen-specific T cell induction, antibody production induction, or immune enhancement, as an antigen mixture Use. The dosage forms of these antigen-specific T cell inducer, antibody production inducer, immunopotentiator and antigen mixture are not particularly limited and may be any of liquid, emulsion, gel, solid, dry powder and the like. In addition to HSV, pharmacologically acceptable carriers and additives such as emulsifiers, surfactants, preservatives and incomplete adjuvants may be contained. For example, HSV may be mixed in advance with Freund's incomplete adjuvant to form an antigen-specific T cell inducer, antibody production inducer, or immune enhancer, and may be mixed with antigen to form an antigen mixture. Further, a plurality of types of antigens in the antigen mixture may be contained.

  The antigen to be used in the present invention is not particularly limited as long as it is other than HSV (including antigens derived from HSV), and may be anything as long as it finally has antigenicity in vivo. Cancer antigens (such as cancer-specific proteins and peptides that are part of them), mycoplasma, viruses (such as AIDS virus, influenza virus, SARS (severe acute respiratory syndrome) coronavirus), bacteria (pathogenicity) Inactivated pathogens and parts of them (such as Escherichia coli, Chlorobacterium, Mycobacterium tuberculosis, etc.), protozoa (malaria, toxoplasma, etc.), parasites (nematodes, etc.) Using pathogen antigens such as cell walls, polysaccharides, bacterial cell components such as proteins, and proteins that constitute viruses) It is possible to make a down. That is, the antigenicity of the vaccine can be enhanced by incorporating the antigen-specific T cell inducer, the antibody production inducer, or the immune enhancer in a vaccine and using it together with a cancer antigen or a pathogen antigen. Here, the vaccine is a live vaccine (attenuated vaccine), killed vaccine, inactivated vaccine, toxoid, component vaccine (component vaccine), genetically modified vaccine (including peptide vaccine, etc.), cancer vaccine, etc. A vaccine with relatively weak antigenicity such as an inactivated vaccine, a component vaccine, a genetically modified vaccine, or a cancer vaccine is preferable. Moreover, it is good also as a mixed vaccine containing multiple types of antigens in a vaccine.

== Inoculation method of antigen mixture ==
The antigen-specific T cell inducer, antibody production inducer, or antigen mixture of the immunopotentiator and antigen thus prepared is inoculated into the living body. The method for inoculating the antigen mixture is not particularly limited, and it may be administered orally or parenterally. Parenteral administration includes subcutaneous, intramuscular, intraperitoneal, intravenous injection, rectal administration, mucosal administration, transdermal administration, or mucosal administration, but is usually inoculated by subcutaneous or intraperitoneal injection There are many cases. Even when the antigen is a cancer antigen, it is not necessary to directly inoculate a tumor having the cancer antigen, and it may be inoculated at a site other than the tumor.

  In addition, the number of inoculations and the place of inoculation are not particularly limited, and may be used in combination with other drugs at the same time. In addition, different antigen mixtures may be inoculated simultaneously.

== Disease treatment methods (including prevention methods) ==
As described above, when an organism is inoculated with an antigen mixture according to the present invention, differentiation and proliferation of antigen-specific T cells added to the antigen mixture and production of antigen-specific antibodies are induced. When the antigen is a cancer antigen or a part of a peptide thereof, typically, antigen-specifically induced T cells attack cancer cells that present the antigen together with histocompatibility antigens on the cell surface. Tumors can be treated with antigen mixtures. In addition, when the antigen is a pathogen antigen, typically, the antigen-specific production-induced antibody attacks the pathogen. Therefore, by inoculating the living body with the antigen mixture prior to infection, Prevention against infection is possible. Moreover, by inoculating a living body with an antigen mixture even after infection, it is possible to treat pathogen infection or prevent recurrence of pathogen infection.

  The principle of the disease treatment method for the antigen to be inoculated is not limited to the case described here. For example, when the tumor is prevented by the method of the present invention or the cancer antigen is a cell surface antigen, it is produced. Tumors may be treated with the induced antibodies.

<Example 1> Cancer vaccine using GARC-1 peptide (1) Preparation of HSV Using KOS strain, which is a wild strain of HSV type I, as HSV, infecting Vero cells according to a conventional method, the culture supernatant The virus released in was collected. As the medium at this time, a serum-free medium (DMEM not containing serum) was used. After measuring the virus concentration, the virus was completely inactivated by either UV treatment for 15 minutes and heat treatment for 30 minutes at 56 ° C, or treatment with 0.1% formalin for 1 week at 4 ° C, It was used for the following experiment (hereinafter, the virus concentration of HSV indicates the value before inactivation). The culture supernatant of Vero cells without virus was treated in the same manner as above and used as a control (Mock).

(2) Vaccine using GARC-1 antigen (tumor antigen) Since mouse glioma cell line GL261 (derived from C57BL / 6 mouse) contains a mutation in GARC-1 gene that does not exist in normal cells, this mutation (AALLNKLYA, SEQ ID NO: 1) (presented on H-2D ^b which is MHC class I) is recognized by T cells as a tumor-specific antigen. Therefore, using this GARC-1 mutant peptide as an antigen used in this example, and using as a control a GARC-1 wild-type peptide (AALLDKLYA, SEQ ID NO: 2) present in normal cells, production of a cancer vaccine Went.

The vaccine
(1) 10 μl (100 μg) of 10 mg / ml GARC-1 mutant peptide or GARC-1 wild type peptide,
(2) 50 μl of a solution containing or not containing inactivated HSV (equivalent to 2 × 10 ⁸ pfu) (Mock),
(3) 40 μl of PBS,
Four types of mixed solutions (100 μl) were prepared for each combination and prepared by mixing with an equal amount of incomplete Freund's adjuvant (BACTO ADJUVANT INCOMPLETE FREUND, DIFCO: 263910) (100 μl) (total) 200 μl).

  A vaccine (200 μl) was inoculated into the skin of both hands and feet of C57BL / 6 mice, a mouse strain from which GL261 was derived, and the same vaccines were again inoculated after 5 days.

Two weeks after the start of vaccine administration, mixed floating cells obtained from the lymph nodes and spleens of mice were adjusted to 2 × 10 ⁶ cells / ml. On the other hand, GL261 cells were treated with mitomycin C (200 μg / ml) for 60 minutes, adjusted to 3 × 10 ⁵ cells / ml, and used as stimulator cells. 1 ml each of these mixed floating cells and stimulating cells were mixed and co-cultured in a 24-well plate (142575 NUNC) for 1 week.

  Thereafter, in the following three experimental systems, analysis of antigen-specific reactivity of T cells (IFN-γ release amount analysis) was performed.

[1. Lymphocyte reactivity to cells presenting GARC-1 peptide (mutant and wild type)]
As described above, the obtained mixed floating cells co-cultured for 1 week were adjusted to 1 × 10 ⁶ cells / ml to obtain effector cells. On the other hand, 293TK ^b D ^b cells (293T cells expressing MHC class I molecules K ^b and D ^b ) were treated with a pulse treatment of GARC-1 mutant peptide or GARC-1 wild type peptide (each in cell culture medium). The peptide was added at a concentration of 10 μg / ml, cultured for 1 hour, washed twice with PBS), adjusted to 1 × 10 ⁶ cells / ml, and used as target cells. These effector cells and target cells were mixed in an amount of 100 μl and co-cultured in a 96-well plate (167008 NUNC) for 2 days. Thereafter, the amount of IFN-γ released in the culture supernatant was analyzed using ELISA kit (KM-IFNG Endogen).

  As a result, as shown in FIG. 1, cell-specific expression of GARC-1 mutant peptide from the lymph nodes and spleens of mice administered with a vaccine containing GARC-1 mutant peptide, which is a tumor antigen, and mock. T cells that produce IFN-γ were induced in response to vaccination with GARC-1 mutant peptide and inactivated HSV as compared to vaccine administration containing this GARC-1 mutant peptide and mock. Mice administered with the vaccine containing enhanced the reactivity of cell-specific lymphocytes presenting GARC-1 mutant peptides. On the other hand, in the control, even when inactivated HSV was added by the vaccine using the self-antigen GARC-1 wild type peptide, it reacted specifically with cells presenting the GARC-1 wild type peptide and caused IFN-γ to react. Producing lymphocytes were not induced.

  From the above, HSV as an adjuvant enhances antigen-specific immunity against tumor antigens, but it does not show a clear immune-enhancing effect against self-antigens and is also useful from a safety standpoint. It became. Therefore, it was concluded that a composition containing HSV is useful as an immunopotentiator against an antigen administered to a living body.

[2. Lymphocyte reactivity I to GL261 cells I]
As described above, the obtained mixed floating cells co-cultured for 1 week were adjusted to 1 × 10 ⁶ cells / ml to obtain effector cells. On the other hand, GL261 cells and control EL4 cells were treated with mitomycin C (200 μg / ml) for 60 minutes, adjusted to 1 × 10 ⁶ cells / ml, and used as target cells. These effector cells and target cells were mixed in an amount of 100 μl and co-cultured in a 96-well plate (167008 NUNC) for 2 days. Thereafter, the amount of IFN-γ released in the culture supernatant was analyzed using an ELISA kit (KM-IFNG Endogen).

  As a result, as shown in FIG. 2, in mice administered with the GARC-1 mutant peptide, which is a tumor antigen of GL261 glioma cells, specific to GL261 cells only when a vaccine containing inactivated HSV was administered. In response, lymphocytes producing IFN-γ were induced.

  From the above, it was shown that by using HSV as an adjuvant in addition to a tumor antigen, immunity specific to tumor cells expressing the tumor antigen can be enhanced. Therefore, it was concluded that inclusion of HSV in the vaccine is useful for enhancing tumor immunity and preventing pathogen infection.

[3. Lymphocyte reactivity II to GL-261 cells]
As described above, CD8 positive T cells (mainly cytotoxic T cells) were purified from the mixed suspension cells obtained by co-culturing for 1 week, and then adjusted to 5 × 10 ⁵ cells / ml to obtain effector cells. On the other hand, GL261 cells were heat-treated at 56 ° C. for 30 minutes, adjusted to 5 × 10 ⁵ cells / ml, and used as target cells. These effector cells and target cells were mixed in an amount of 100 μl and co-cultured in a 96-well plate (167008 NUNC) for 1 day. Thereafter, the amount of IFN-γ released in the culture supernatant was analyzed using an ELISA kit (KM-IFNG Endogen).

  As a result, as shown in FIG. 3, when a mouse was immunized with a vaccine containing GARC-1 mutant peptide that is a tumor antigen of GL261 glioma cells and HSV, each was compared with a mouse immunized alone. Thus, the reactivity of CD8 positive T cells producing IFN-γ with respect to GL261 cells was enhanced (FIG. 3).

  From the above, it was shown that the induction of CD8 positive T cells responding to tumors was enhanced by using HSV as an adjuvant in addition to tumor antigens. Therefore, it was concluded that the immunopotentiator containing HSV is useful as an antigen-specific T cell inducer for the antigen in combination with HSV.

<Example 2> Cancer vaccine HSV using AH1 peptide (tumor antigen) was prepared in the same manner as Example 1.
The antigen is a tumor antigen of mouse colon cancer cell line CT26 (derived from BALB / c mouse), and peptide AH1 (SPSYVYHQF, SEQ ID NO: 3) presented on MHC class I (H-2L ^d ) (Huang A et al.) al., Proc. Natl. Acad. Sci. USA 93. 9730-9735, 1996), and as a control peptide presented on the same MHC class I (H-2L ^d ), tumor antigen of mouse mastocytoma cell line P815 The peptide P815A (LPYLGWLVF, SEQ ID NO: 4) (Lethe B, et al, Eur. J. Immunol 22, 1992) was used.

The vaccine
(1) AH1 peptide (100 μg) or P815A peptide (100 μg)
(2) Inactivated HSV (equivalent to 2 × 10 ⁸ pfu) or Mock
(3) PBS
Four kinds of the mixture (100 μl) were prepared for each combination and mixed with an equal amount of incomplete Freund's adjuvant (100 μl) (total 200 μl). Vaccine (200 μl) was administered into both hands and feet skin of BALB / c mice, which is a mouse strain derived from CT26, and the same vaccines were administered again 7 days later. One week after the second vaccine administration, 5 × 10 ⁵ CT26 tumor cells were transplanted on the back of BALB / c mice, and the tumor diameter was measured over time to analyze the tumor volume. The results are shown in Table 1.

  In mice administered with vaccines of P815A + HSV (3 individuals) and P815A + Mock (4 individuals), the engraftment of CT26 tumors was confirmed in all cases on the 8th day after tumor implantation, after which the tumor volume increased and was rejected. It never happened. On the other hand, in the mice (4 individuals) administered with the vaccine of AH1 + Mock, the engraftment of CT26 tumor was confirmed in all cases on the 8th day after the tumor transplantation, but the tumor was subsequently reduced, and on the 22nd day after the tumor transplantation, Tumors were rejected in 3 cases except 1 case. Furthermore, in the mice (3 individuals) administered with the vaccine of AH1 + HSV, tumor engraftment was not observed within the observation period, and all cases were completely rejected.

  From the above, HSV was shown to actually enhance the antigen-specific immune response of colorectal cancer cell CT26, and its usefulness as an adjuvant for enhancing the effect as a cancer vaccine was shown. Therefore, it was concluded that a vaccine containing an immunopotentiator containing HSV is effective as a cancer vaccine.

<Example 3> Treatment of tumor using AH1 peptide (tumor antigen) HSV was purified in the same manner as in Example 1. As the tumor antigen, AH1 peptide was used as in Example 2, CT26 (mouse colon cancer) was used as the target tumor cell, and Meth A (mouse fibrosarcoma) was used as the control tumor cell.

First, CT26 cells (5 × 10 ⁵ cells) were transplanted into the dorsal part of Balb / c mice, and the first immunization was started on the fifth day of transplantation.

For the first immunization,
(1) AH1 peptide (50 μg) or DMSO
(2) Inactivated HSV (equivalent to 1 × 10 ⁸ Pfu) or Mock
(3) PBS
3 types (AH1 / HSV, and AH1 / Mock and DMSO / Mock as negative controls) were prepared and mixed with an equal volume of incomplete Freund's adjuvant (IFA) (100 μl, respectively) HSV (IFA), and AH1 / IFA and DMSO / IFA as negative controls; 200 μl each). As a positive control, a mixture of AH1 peptide and PBS (100 μl) was mixed with an equal amount of complete Freund's adjuvant (CFA) (100 μl) (denoted as AH1 / CFA; 200 μl). The antigen thus prepared (100 μl each) was administered to both foot pads of Balb / c mice.

For the second immunization 7 days later,
(1) AH1 peptide (for AH1 / HSV (IFA), AH1 / IFA, and AH1 / CFA) or DMSO (for DMSO / IFA)
(2) PBS
(100 μl) was prepared for each combination, mixed with an equal volume of incomplete Freund's adjuvant IFA (100 μl) (total 200 μl), and the antigens (100 μl each) were administered to the foot pads in the same manner.

  Thereafter, antigen-specific reactivity of T cells was analyzed in the following three experimental systems.

1. Evaluation of anti-tumor effect The volume of the tumor transplanted into the mice was measured every 2 days until 14 days and every 4 days until 21 days, and the immune effect was evaluated by the change in the tumor volume. FIG. 4A shows the experimental results for each mouse, and FIG. 4B shows the average value of the tumor volume on the 21st day.

  From the time course of the tumor volume (FIG. 4A), the increase in tumor volume was suppressed when AH1 / CFA (positive control) was used, compared with the case where DMSO / IFA and AH1 / IFA were used. When / HSV is used, it turns out that the increase in the tumor volume is further suppressed. In addition, when comparing the average tumor volume on day 21 (FIG. 4B), the tumor volume was not statistically significant when AH1 / CFA was used compared to when DMSO / IFA and AH1 / IFA were used. On the other hand, when AH1 / HSV was used, an increase in tumor volume was significantly suppressed (p <0.05).

  Thus, it became clear that HSV showed the inhibitory effect of the tumor growth of colon cancer cell CT26 by administering with AH1.

2. Evaluation of Cytotoxic Activity 21 days after tumor cell transplantation, mixed floating cells were isolated from the spleen and lymph nodes of each mouse and prepared to 3 to 5 × 10 ⁶ / ml. On the other hand, as antigen-presenting cells, spleen cells prepared from normal mice were irradiated with X-rays (60 Gy) and reacted with peptide antigen AH1 (10 μM) for 90 minutes to prepare 1 × 10 ⁶ / ml. 1 ml of each of these mixed floating cells and antigen-presenting cells was mixed and co-cultured in a 24-well plate (NUNC 142475) for 5 days. Thereafter, CD8 positive T cells (E: T-ratio: 60, 30, 15; 3 × 10 ⁵ , 1.5 × 10 ⁵ , 0.75 × 10 ⁵ ) purified from co-cultured floating cells were transferred to ⁵¹ Cr ( Amersham) -labeled CT26 cells (5 × 10 ³ cells) were mixed and co-cultured in 96-well plates (U-bottom: BD35 3077) for 4 hours, and cytotoxic activity was evaluated by ⁵¹ Cr release assay. did. In addition, Meth A cells (5 × 10 ³ cells) were used as control cells. CT26 cells were loaded with 1.85 MBq of ⁵¹ Cr in 1 ml of cell suspension (1 × 10 ⁶ cells) and shaken at 37 ° C. for 1-2 hours.

  As a result, as shown in FIG. 4C, when AH1 / HSV was used, the specific cytotoxicity against CT26 cells was significantly enhanced (p <0.05) compared to when DMSO / IFA and AH1 / IFA were used. It was.

  Thus, HSV strongly induces antigen-specific CD8-positive T cells (mainly considered to be cytotoxic T cells) and enhances cytotoxic activity against CT26 cells when administered together with the AH1 peptide. It was shown that.

3. Evaluation of cytokine IFN-γ production
Using BD Cytofix / Cytoperm kit (Pharmingen), IFN-γ producing cells in the mixed suspension cells co-cultured as described above were analyzed.

  First, in order to inhibit intracellular protein transport for co-cultured mixed floating cells, 1.5 μl / 2 ml of BD GolgiStop containing monensin was added to the wells of a 24-well plate, and after 8 hours, nonspecific reactions were suppressed. In order to achieve this, an antibody Fc-Block against Fc receptor (mAb 2.4G2: diluted 100-fold) was added, and FITC-labeled anti-CD4 antibody (mAb GK1.5 e-Bioscience diluted 400-fold) or FITC-labeled anti-CD8 antibody ( mAb 53-6.7 e-Bioscience, diluted 400-fold). Thereafter, 100 μl of BD Cytofix / Cytoperm solution was added, the cells were fixed at 4 ° C. for 20 minutes, washed twice with BD Perm / Wash solution, and PE (phycoerythrin) -labeled anti-IFN-γ antibody (mAb XMG1.2: FACS analysis was carried out with 50 μl of BD Perm / Wash solution containing e-Bioscience (200-fold dilution) for 30 minutes at 4 ° C.

  As a result, it was found that in the AH1 / HSV immunized group, most of the IFN-γ producing cells increased by AH1 peptide co-culture were not CD4-positive T cells but CD8-positive T cells (FIG. 4D).

  From the above, it was shown that cancer antigen-specific CD8-positive T cells are strongly induced and cancer immunity is enhanced by using HSV as an adjuvant together with a cancer antigen in the treatment of a tumor using a cancer antigen. Therefore, it was concluded that a vaccine containing an immunopotentiator containing HSV together with a cancer antigen is effective for treating a tumor using the cancer antigen.

<Example 4> Influenza virus vaccine HSV using hemagglutinin antigen (HA) was purified in the same manner as in Example 1. The animals used here are Balb / c mice (／: n = 4 / group).

For the first immunization, hemagglutinin antigen protein (HA) (H1N1: IFA-055-B0522) (10 μg) was used,
(1) HA (10 μg / 10 μl) or DMSO (10 μl)
(2) Inactivated HSV (low): 1 × 10 ⁷ Pfu, Inactivated HSV (high): 1 × 10 ⁸ Pfu, or Mock
(3) PBS
The following 5 types of liquid mixture (100 μl) were prepared for each combination, mixed with an equal volume of incomplete Freund's adjuvant (IFA) (100 μl) (total 200 μl), and added to the foot pads of both feet of Balb / c mice. Vaccine (100 μl each) was administered.
(1) DMSO / IFA
(2) DMSO / HSV
(3) HA / IFA
(4) HA / HSV (low)
(5) HA / HSV (high)

The second immunization, for the purpose of enhancing the immune response against CD4 epitopes or CD8 epitope of hemagglutinin antigen, a peptide antigen, ^{I-E d} restricted HA peptide (SVSSFERFEIFPK SEQ ID NO: 5, corresponding to amino acid numbers 124-136) And a ^Kd- restricted HA peptide (IYSTVASSL SEQ ID NO: 6, corresponding to amino acid numbers 533-541) (50 μg each) were used. That is,
The second vaccine after 7 days
(1) IE ^d- restricted HA peptide (50 μg) and K ^d- restricted HA peptide (50 μg), or DMSO
(2) PBS
(50 μl) was mixed with an equal volume of incomplete Freund's adjuvant (50 μl) (100 μl), and vaccines (50 μl each) were administered to both footpads of Balb / c mice as shown below for each combination. did.
(1) DMSO / IFA
(2) to (5) HA peptide / IFA

  Thereafter, antigen-specific reactivity of T cells was analyzed in the following two experimental systems, and the antibody titer of the induced antibody was measured.

1. Evaluation of cytotoxic activity One week after the second vaccine administration, mixed floating cells were isolated from the spleen and lymph nodes of each mouse and prepared to 3 to 5 × 10 ⁶ / ml. On the other hand, spleen cells prepared from normal mice as antigen-presenting cells were irradiated with X-rays (60 Gy), treated with IE ^d- restricted HA peptide or K ^d- restricted HA peptide (10 μM) for 90 minutes, and 1 × Prepared to 10 ⁶ / ml. 1 ml of each of these mixed floating cells and antigen-presenting cells was mixed and co-cultured in a 24-well plate (NUNC 142475) for 5 days. Thereafter, CD8 positive T cells (E: T-ratio: 60, 30, 15; 3 × 10 ⁵ , 1.5 × 10 ⁵ , 0.75 × 10 ⁵ ) purified from co-cultured floating cells were obtained with ⁵¹ Cr. After mixing with CT26 cells (treated with 10 μM ^Kd- restricted HA peptide) (5 × 10 ³ cells) labeled as described above and co-cultured in 96-well plates (U-bottom: BD35 3077) for 4 hours Cytotoxic activity was assessed by a ⁵¹ Cr release assay. As control cells, CT26 cells (treated with 10 μM P815A peptide) (5 × 10 ³ cells) were used.

As a result, as shown in FIG. 5A, (1) DMSO / IFA, DMSO / IFA, (3) HA / IFA, HA peptide / IFA, compared with the combination of (5) HA / HSV (high), HA peptide / IFA combination significantly (p <0.05) strongly induces antigen-specific CD8 positive T cells and is shown to enhance cytotoxic activity against CT26 cells (treated with 10 μM ^Kd- restricted HA peptide) It was done.

2. CD4-positive T cells purified from a mixed suspension cells was assessed cocultures proliferative response (1 × 10 ⁵⁾ were treated with the antigen-presenting cells ^{(I-E d} restricted HA peptides after X-ray irradiation (60 Gy) Spleen cells) (5 × 10 ⁴ cells or 1 × 10 ⁵ cells) and co-culture for 3 days. Add [ ³ H] -thymidine (18.5 kBq / well) to the medium 5 hours before the end of the culture to complete the culture. Thereafter, the proliferation response was evaluated by the amount of uptake (count value cpm of [ ³ H]).

  As shown in FIG. 5B, the results were as follows: (1) CD4 positive T cells of mice immunized with a combination of DMSO / IFA and DMSO / IFA. (4) Combination of HA / HSV (low) and HA peptide / IFA And (5) CD4-positive T cells of mice immunized with the combination of HA / HSV (high) and HA peptide / IFA showed a significant (p <0.05) proliferation promoting effect depending on the number of antigen presenting cells.

  Thus, when administering influenza vaccine, it became clear that antigen-specific CD4-positive T cells can be induced by using HSV as an adjuvant together with influenza antigen.

3. Measurement of antibody titer The antibody titer was measured for 4 mice each of (1) to (4) by collecting sera from peripheral blood one week after the second vaccine, and measuring it by hemagglutination inhibition test (HI).

  Specifically, the antibody titer against influenza A virus HA antigen was examined using a kit of influenza virus HI reagent “Seiken” (Denka Seiken Co., Ltd.). Add 0.1 ml of RDE (Receptor Destroying Enzyme) to 0.1 ml of the sample, and remove the non-specific inhibitor by culturing at 37 ° C. overnight, and stop the reaction of RDE treatment by heating at 56 ° C. for 30 minutes. 0.6 ml of brine was added. 50 μl of 50% (v / v) chicken erythrocyte suspension was added to the RDE-treated sample, mixed, allowed to stand at 4 ° C. for 60 minutes, centrifuged, and the supernatant was started from 1:10 to make serial dilutions. . HA antigen was added to each of them and mixed thoroughly, and then 0.5% (v / v) chicken erythrocyte suspension was added to examine how many times the hemagglutination was suppressed and the HI antibody titer was measured.

As a result of this hemagglutination inhibition test, the HI antibody titer was less than 10 in both (1) DMSO / IFA, DMSO / IFA combination and (2) DMSO / HSV, HA peptide / IFA combination. On the other hand, (3) HI antibody titer was detected by the combination of HA / IFA and HA peptide / IFA (65 ± 64.03), but (4) HI was detected by the combination of HA / HSV (low) and HA peptide / IFA. The antibody titer increased significantly (p <0.05) (140 ± 40). (However, the values in parentheses are mean ± standard deviation)
From this result, it became clear that the serum antibody titer against the influenza antigen was significantly increased by simultaneously administering HSV as an adjuvant together with the influenza antigen.

4). Conclusion From the above, HSV was shown to enhance the antigen-specific immune response of influenza antigens, and it was shown to be useful as an adjuvant to enhance the effect as an influenza vaccine. Therefore, it was concluded that a vaccine containing an immunopotentiator containing HSV together with an influenza antigen is effective as an influenza vaccine.

According to the present invention, it has become possible to provide a novel immune enhancer containing a herpes simplex virus, a novel antibody production inducer, and a vaccine containing them.

Claims (26)

  An antigen-specific T cell inducer for an antigen other than HSV, which contains herpes simplex virus (HSV) as an active ingredient.   2. The antigen-specific T cell inducer according to claim 1, which is an antigen-specific cytotoxic T cell inducer   3. The antigen-specific T cell inducer according to claim 1 or 2, wherein the herpes simplex virus is inactivated.   The antigen-specific T cell inducer according to claim 3, wherein the herpes simplex virus is inactivated by heating, UV irradiation, irradiation, fixation, or genetic recombination.   The antigen-specific T cell inducer according to any one of claims 1 to 4, further comprising an incomplete Freund adjuvant (IFA).   An agent for inducing antibody production against an antigen other than HSV, which contains herpes simplex virus (HSV) as an active ingredient.   The antibody production inducer according to claim 6, which is an antigen-specific cytotoxic T cell inducer.   The antibody production inducer according to claim 6 or 7, wherein the herpes simplex virus is inactivated.   The antibody production-inducing agent according to claim 8, wherein the herpes simplex virus is inactivated by heating, UV irradiation, radiation irradiation, fixation, or genetic recombination.   The antibody production inducer according to any one of claims 6 to 9, further comprising an incomplete Freund adjuvant (IFA).   An immunopotentiator for antigens other than HSV, which contains herpes simplex virus (HSV) as an active ingredient.   The immunopotentiator according to claim 11, wherein the herpes simplex virus is inactivated.   The immunopotentiator according to claim 12, wherein the herpes simplex virus is inactivated by heating, UV irradiation, irradiation, fixation, or genetic recombination.   The immunopotentiator according to any one of claims 11 to 13, further comprising an incomplete Freund adjuvant (IFA). A vaccine against antigens other than herpes simplex virus (HSV),
An immunopotentiator comprising herpes simplex virus as an active ingredient and a vaccine containing the antigen.   The vaccine according to claim 15, wherein the herpes simplex virus is inactivated.   The vaccine according to claim 16, wherein the herpes simplex virus is inactivated by heating, UV irradiation, irradiation, fixation, or genetic recombination. The vaccine according to any one of claims 15 to 17, further comprising an incomplete Freund adjuvant (IFA).   The vaccine according to any one of claims 15 to 18, which is a cancer vaccine.   The vaccine according to any one of claims 15 to 18, which is a preventive vaccine against infection with a pathogen. A method of inducing antigen-specific T cells to an antigen other than HSV administered to a vertebrate,
A method comprising administering the antigen-specific T cell inducer according to any one of claims 1 to 5 or the vaccine according to any one of claims 15 to 20 in combination with the antigen.   The method of claim 21, wherein the T cell is a cytotoxic T cell. A method for inducing antigen-specific antibody production against an antigen other than HSV administered to a vertebrate,
A method comprising administering the antibody production inducer according to any one of claims 6 to 10 or the vaccine according to any one of claims 15 to 20 in combination with the antigen. A method of enhancing immunity in vertebrates other than humans,
A method comprising administering the immunopotentiator according to any one of claims 11 to 14, or the vaccine according to any one of claims 15 to 20. A method of treating tumors in vertebrates other than humans,
A method comprising inoculating the vaccine according to any one of claims 15 to 19. A method for treating pathogen infections in non-human vertebrates,
A method comprising inoculating the vaccine according to any one of claims 15 to 18, 20.

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